Effect of MnxOy Nanoparticles Stabilized with Methionine on Germination of Barley Seeds (Hordeum vulgare L.)

The aim of this research was to study the effect of MnxOy nanoparticles stabilized with L-methionine on the morphofunctional characteristics of the barley (Hordeum vulgare L.) crop. MnxOy nanoparticles stabilized with L-methionine were synthesized using potassium permanganate and L-methionine. We established that MnxOy nanoparticles have a diameter of 15 to 30 nm. According to quantum chemical modeling and IR spectroscopy, it is shown that the interaction of MnxOy nanoparticles with L-methionine occurs through the amino group. It is found that MnxOy nanoparticles stabilized with L-methionine have positive effects on the roots and seedling length, as well as the seed germination energy. The effect of MnxOy nanoparticles on Hordeum vulgare L. seeds is nonlinear. At a concentration of 0.05 mg/mL, there was a statistically significant increase in the length of seedlings by 68% compared to the control group. We found that the root lengths of samples treated with MnxOy nanoparticle sols with a concentration of 0.05 mg/mL were 62.8%, 32.7%, and 158.9% higher compared to samples treated with L-methionine, KMnO4, and the control sample, respectively. We have shown that at a concentration of 0.05 mg/mL, the germination energy of seeds increases by 50.0% compared to the control sample, by 10.0% compared to the samples treated with L-methionine, and by 13.8% compared to the samples treated with KMnO4.

Another important area of application of nanomaterials, in particular, manganese nanoparticles and their oxides, is agriculture [31][32][33]. A large number of research papers are devoted to the study of the influence of nanoscale oxide forms on the morphofunctional characteristics of agricultural plants [34][35][36][37][38][39][40][41][42][43][44][45][46][47][48]. For example, the work of Margenot et al. [49] shows that the addition of copper oxide (CuO) nanoparticles increases the diameter of the roots of lettuce seedlings by 52% and the diameter of carrot seedlings by 26%. However, another form of this microelement did not show any significant effect. The effect of zinc oxide (ZnO) nanoparticles on the growth and antioxidant activity of chickpea seeding of the Cicer arietinum L. var. HC-1 culture was studied by Burman et al. [50]. It is shown that the addition of ZnO nanoparticles has a positive effect on the dry weight of shoots in comparison to the control sample microparticles of the inorganic form of zinc (ZnSO 4 ), at a concentration of 1.5 ppm.
According to the FAO, there are about 1750 gene banks (seed banks) in the world, containing a total of 7.4 million agricultural seed samples [51]. These seeds have been stored for more than 10 years. At the same time, a number of studies have shown that these seeds have reduced germination energy and other morphofunctional characteristics during storage. For example, Maryam et al. proved that the germination percentage, germination energy, dry weight of seedlings and seed weight decrease significantly as seeds age [52]. Walters et al. studied wild and cultivated species from the collections of the gene bank of the US Department of Agriculture [53]. The study included 276 different species (codes W1-W276) with a total of 41,286 specimens collected from 1963 to 1968. The seeds were stored for 30-60 years, until 1977, at a temperature of +5 • C, and after that at −18 • C. The initial germination percentage, the actual germination percentage, and the number of years of storage were recorded and the half-life was calculated. The authors found that the germination percentage of old seeds averages from 40% to 60%.
Nanoparticles can be used to solve this problem. Younes et al. described the effect of silver nanoparticles (Ag NPs) on the germination of seven-year-old Vicia faba seeds [54]. The authors investigated the genotoxic effects associated with seed aging and were able to achieve an increase in root length and germination percentage of Vicia faba seeds. It is shown that when seeds are treated with 10 pm Ag NPs, there is an increase in the viability of seedlings of Vicia faba seeds. Yang et al. showed that titanium dioxide nanoparticles significantly increase the viability of old seeds and the formation of chlorophyll, stimulate the activity of ribulose 1,5-bisphosphate carboxylase, and enhance photosynthesis, thereby accelerating the growth and development of plants [55].
It is known that manganese-containing nanoparticles can be used in agriculture as fertilizers and for plant stress management [38,[56][57][58][59][60]. Pradhan et al. [59] noted that manganese nanoparticles are the best source of the trace element Mn and have less toxicity to the Vigna radiata culture compared to the inorganic form (MnSO 4 ). The use of Mn nanoparticles leads to an increase in the length of roots and sprouts by 40-70% compared to seeds treated with MnSO 4 solution. Ye et al. [61] evaluated the effect of manganese nanoparticles on the germination of Capsicum annuum L. seeds in conditions of salt stress. It has been established that under salt stress, manganese nanoparticles behave as an important factor in the control of oxidative stress, contributing to the redistribution of trace elements between roots and shoots. Kasote et al. found that manganese oxide nanoparticles have lower phytotoxicity compared to other manganese-containing forms (KMnO 4 and Mn 2 O 3 ), change the chlorophyll and antioxidant profiles of seedlings, and had a noticeable effect on phenolic acids and phytohormones of watermelon seedlings at a concentration of 20 mg/L [62].
The purpose of this work was the synthesis and characterization of Mn x O y nanoparticles stabilized with L-methionine, as well as the study of their effect on the morphofunctional characteristics of agricultural barley (Hordeum vulgare L.) seeds stored for more than 10 years. The choice of seeds with reduced morphofunctional characteristics is also due to the presence of a large amount of literature that uses standard seeds with normative and permissible morphofunctional characteristics to study the phytotoxicity of manganese compounds. However, at low manganese concentrations, as shown by Kasote et al. [62], Varaprasad et al. [63], and Wali et al. [64], toxic properties are not exhibited and metabolic processes are not normalized, being the main part of many manganese-containing enzymes, thus improving the morphofunctional parameters of seeds and young plants.

Materials and Methods
Reagent grade chemicals and grade A glassware were used in the present study. Conductivity of distilled water used was less than 1 µS/cm. All experiments were carried out in three-fold repetitions.

Synthesis of Mn x O y Nanoparticles
The synthesis of nanoscale Mn x O y stabilized with methionine was carried out according to the method presented in previous work [65]. Potassium permanganate, methionine, and distilled water were used for the synthesis. First, 0.005 g of L-methionine was weighed on analytical scales and put into 200 mL flask with distilled water. The resulting solution was stirred for a minute on an electromagnetic stirrer. In the second stage, the same process was completed with 0.005 g of potassium permanganate. At the third stage, two prepared solutions were mixed and stirred for 15 min. To study the effect of Mn x O y nanoparticles on the seeds, only freshly prepared solutions were used. For research using diffractometry and scanning electron microscopy, the Mn x O y samples were centrifuged at 15,000 rpm for 10 min and dried at 50 • C (2 h).

Samples Measurement
Hydrodynamic radius of synthesized Mn x O y stabilized with L-methionine was measured with photon correlation spectrometer Photocor Complex (Photocor, Moscow, Russia).
The morphology of Mn x O y particles was studied using a Mira-LMH scanning electron microscope (Tescan, Brno, Czech Republic). On a standard instrument table (12 mm), a double-sided conductive carbon tape was set. A thoroughly mixed Mn x O y sample was taken from a dry powder ground in a mortar and applied to the tape. Then, carbon deposition of the order of 10 nm was performed on the QR 150 sputtering system and gas (nitrogen) was introduced into the microscope system by opening the balloon.
X-ray powder diffraction (XRD) analysis was conducted using X-ray diffractometer Empyrean (PANalytical, Almeo, The Netherlands) to determine the crystalline structure of nanoparticles.
Parameters of the measurement: The determination of the gelation point was carried out on a rotary viscometer "Fungilab Expert" ("Fungilab S. A.", Barselona, Spain), the action of which is based on the use of viscous friction arising in a layer of liquid flowing in the annular gap between the rotating and stationary cylinders.
Parameters of the measurement: The rotation speed of 0.1-200 rpm [66].
The molecular simulation was carried out in the IQmol molecular editor, and the quantum-chemical calculations of the models were carried out using the QChem software with the following parameters: Calculation-Energy, method-B3LYP, Basis-6-31G*, Convergence-5, Force field-Chemical [67].
To study functional groups in the obtained samples, IR spectroscopy was used. IR spectra were recorded on an FSM-1201 IR-spectrometer with Fourier transform. The measurement range was 400-4400 cm −1 .
The ζ-potential and active acidity of the medium was determined by acoustic and electroacoustic spectroscopy on a DT-1202 setup (Dispersion Technology, NY, USA) [68]. The method of preparation of solutions with an accurate pH is presented in the previous published work [69].

Treatment of the Hordeum vulgare L. Seeds
In the experiment, seeds of the Hordeum vulgare L. culture used were stored for more than 10 years, with reduced morphological and functional characteristics. The seeds of the Hordeum vulgare L. culture were collected and identified on the basis of the Stavropol Research Institute of Agriculture (SNIISH, Stavropol, Russian) in 2011. The samples were stored in the biological collection of grain ears, sorghum crops, perennial fodder, and medicinal plants at the Stavropol Research Institute of Agriculture. Seed collection was carried out according to Russian national standard GOST 12036-85 "Seeds of agricultural crops. Acceptance rules and sampling methods". Appropriate permission has been obtained for the collection of seeds samples.
Previously, the seeds were not subjected to further processing. The seeds were treated with solutions of Mn x O y stabilized with L-methionine, L-methionine, and potassium permanganate (KMnO 4 ) at various concentrations (0.0005; 0.005; 0.05; 0.5; 5 mg/mL). In the control sample, the seeds were treated with distilled water.
Hordeum vulgare L. seeds treated with solutions of various concentrations were placed in a thermostat at a constant temperature of 20 • C without illumination. Each sample contained 100 seeds. A total of 72 h after treatment, the percentage of germinated seeds (germination energy) was calculated, lengths of root and seedling were measured, and the obtained data were compared with the control.

Statistical Data Processing
Statistical processing of the experimental results was carried out using Statistica 12.0 software (StatSoft, Tulsa, OK, USA). To assess the significance of dressing agent's concentration as a factor affecting the seeds germination percentage, the length of roots, and shoots length, intragroup and intergroup analysis of variance (ANOVA) was carried out. The nature of the distribution of quantitative characteristics was determined using the Shapiro-Wilk test, the equality of variances was determined using the Leuven test. Tukey's multiple comparison test was used to determine the significant difference between the samples. To compare quantitative characteristics in the intergroup analysis of variance, the Kruskal-Wallis test was used. Quantitative values were assessed using the median (Me) and the 25th and 75th percentiles. The critical level of significance (p) was taken equal to 0.05.

Mn x O y Nanoparticles Characterization
In the first stage of the research, dried samples of Mn x O y nanoparticles stabilized with L-methionine were examined by scanning electron microscopy (SEM) and powder diffractometry. The data obtained are presented in Figures 1 and 2.  Analysis and interpretation of the diffractogram ( Figure 2) showed the presence of low-intensity broadened peaks indicating the existence of an amorphous MnO 2 phase with a tetragonal crystal lattice having spatial group I4/m (unit cell parameters: a-9.8150 Å, b-9.8150 Å, c-2.8470 Å) [70]. The existence of the second phase Mn 3 O 4 , with a tetragonal crystal lattice having spatial group I41/amd (unit cell parameters: a-5.7574 Å, b-5.7574 Å, c-9.4239 Å) is also established [71].
To study the mechanism of stabilization of Mn x O y nanoparticles stabilized with Lmethionine, quantum chemical modeling was carried out in the QChem software [72][73][74]. According to the literature data [75], methionine is oxidized to methionine sulfoxide ( Figure 3). At the first stage of quantum chemical modeling, the models of methionine sulfoxide, MnO 2 and Mn 3 O 4 were considered separately. The manganese dioxide and double manganese oxide molecules are represented by sequences of three manganese atoms connected to a certain number of oxygen atoms, which correlates with the valencies of these Mn atoms and, taking into account the coordination field, the free bonds are hydrogenated. The obtained models, electron density distributions, electron density distribution gradients, and molecular orbitals are presented in Figures 4-6.    From the data analysis, it was found that the interaction of methionine sulfoxide with manganese oxides can occur through the oxygen of the sulfogroup, the amino group and the carboxyl group. In the next stage of modeling, the variants of the interaction of methionine sulfoxide with manganese oxides are considered. The obtained models and the results of quantum chemical calculations are presented in Table 1 and in the Supplementary (Figures S1-S12). According to references [76][77][78][79], the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) are the main orbitals describing the chemical stability of the system. HOMO is directly related to the concept of "ionization potential". LUMO is related to the concept of "electron affinity". The energy difference between the LUMO and HOMO orbitals is called the energy gap (∆E). Based on the data obtained, the value of the chemical hardness (η) was calculated. The chemical hardness characterizes the stability of the system. The chemical hardness is calculated as follows: where E LUMO -LUMO energy; E HOMO -HOMO energy. The analysis of the obtained data showed that the interaction of methionine sulfoxide with manganese oxides is an energetically advantageous process, as evidenced by a decrease in the total energy (E). The interaction of the amino group of methionine sulfoxide with MnO 2 and Mn 3 O 4 was found to be the most energetically advantageous (Figures 7 and 8).   To confirm the quantum chemical modeling data, IR spectroscopy of the obtained samples of Mn x O y nanoparticles stabilized with L-methionine was performed. The obtained data are presented in Figure 9.  [82,83].
In the IR-spectrum of Mn x O y nanoparticles, bands characteristic of deformation vibrations are observed in the same range from 400 cm −1 to 2200 cm −1 : the range from 439 cm −1 to 779 cm −1 corresponds to vibrations of -CH and -CH 2 bonds, at 880 cm −1 -vibrations of -C-C bonds, the range from 1281 cm −1 to 1341 cm −1 corresponds to fan vibrations of the -CH 2 bond, the range from 1404 cm −1 to 1645 cm −1 corresponds to symmetric vibrations of the ionized amino group NH 3 + , at 1743 cm −1 -asymmetric vibrations of the carboxyl group COO − [84,85]. It is important to note that in the IR spectrum of the sample there are oscillations in the range from 1058 to 1150 cm −1 , which are characteristic of scissor oscillations of the S=O group. The presence of this group confirms the oxidation of L-methionine and the formation of methionine sulfoxide [86,87].
It is important to note that in the IR-spectrum of Mn x O y nanoparticles, there is a decrease in the intensity of the bands at 1341 cm −1 and 1514-1609 cm −1 , which indicates the formation of a chemical bonds and the interaction of Mn x O y nanoparticles with Lmethionine through the amino group [88,89].
Then, information was obtained on the effect of the Mn x O y sol concentration on the gelation process in this colloidal system.
A graph of the dependence of the dynamic viscosity of the nanoscale Mn x O y solutions on its concentration is presented in Figure 10. Analysis of Figure 10 showed that the resulting sample can be characterized as a Newtonian liquid with a gelation point of 0.1 mg/mL. The result is that the dynamic viscosity of solutions of nanoscale Mn x O y in the concentration range from 0.0005 to 0.1 mg/mL did not change significantly and was 1-3 MPa·s. This indicates that at these concentrations, the samples are a free-dispersed colloidal systems (sol). When the concentration of nanoscale Mn x O y is 0.1 mg/mL, a sharp jump in the dynamic viscosity is observed, which indicates that the gelation point has been reached and the type of colloidal system has changed. At a concentration of nanosized Mn x O y ≥ 0.1 mg/mL, bound-dispersed (gel-cohesive) systems are formed, which can be identified as a Newtonian fluid. To confirm the obtained data, the hydrodynamic radius of Mn x O y nanoparticles solutions was studied before the gelation point (0.05 mg/mL) and after (0.5 mg/mL). The resulting histograms are shown in Figures 11 and 12.   Figure 11, the distribution of hydrodynamic particle radii in Mn x O y samples is monomodal with an average value of 25 nm and a dispersion of 10-100 nm. The analysis of Figure 12 showed that the sample has a bimodal distribution and consists of two fractions. The particles of the first fraction have an average hydrodynamic radius of 25 nm and a dispersion of 10-80 nm, while particles of the second fraction have an average hydrodynamic radius of 512 nm and a dispersion of 100-1000 nm. The data obtained are consistent with the works [90,91]. The results obtained are consistent with the SEM microscopy data.

As shown in
At the next stage, the effect of the pH of the solution on the ζ-potential of Mn x O y nanoparticles stabilized with methionine was investigated. The obtained dependence is shown in Figure 13. It is established that during the transition from an acidic medium to an alkaline one, the nanoparticles "recharge" occurs, i.e., the ζ-potential changes from +48.5 mV to −15.5 mV. It is important to note that an isoelectric point is observed at pH = 8.25. The obtained result is consistent with the work of Neves et al. [92]. As is known, an acidic environment leads to the activation of amino groups of amino acids, and an alkaline environment leads to the activation of carboxyl groups of amino acids [93,94]. Upon protonation of an amino group in an acidic medium, the molecule L-methionine acquires a positive charge (NH 3 + ) and interacts with the Mn x O y micelle, as shown above by the simulation results and IR spectroscopy. With a decrease in hydrogen ions concentration (an increase in pH), the speed of the protonation process slows down, and the equilibrium shifts in the opposite direction, and as a result the charge of the amino groups decreases and becomes zero at the isoelectric point. A further increase in the concentration of OH − ions, above the isoelectric point, leads to a change in the charge of the L-methionine molecule, which becomes negative, due to the deprotonization of carboxyl groups. The scheme of the described chemical transformations is shown in Figure 14. Thus, an increase in the active acidity leads to a loss of NH 3 + charge and an increase in the desorption of uncharged L-methionine molecules from the surface of Mn x O y nanoparticles. As a result, the particles lose their stabilizing shell and begin to coagulate, forming the spatial structure of the gel, which is shown in Figure 15.

Investigation of the Effect of Mn x O y Nanoparticles Stabilized with Methionine on Germination of Barley Seeds
The results of the study of the effect of Mn x O y nanoparticles stabilized with methionine on the morphofunctional parameters of seeds germination are presented in Tables 2 and 3 and in the Supplementary (Figures S13-S15). The analysis of the obtained data showed that 10 years' storage has reduced morphofunctional characteristics of barley seeds. It was found that the germination energy of control samples was less than 50%. It was also set that the length of seeding in the concentration range of 0.0005-0.5 mg/mL for Mn x O y nanoparticles, potassium permanganate KMnO 4, and for L-methionine is higher than that of the control sample. Similarly, it is discovered that when the concentration of L-methionine is 5 mg/mL, the length of the Hordeum vulgare L. culture seeding takes values lower values than the values of the control sample. There was a statistically significant prevalence of seeding length in samples treated with 0.05 mg/mL Mn x O y nanoparticles (32.13 ± 3.40 mm) by 68% compared to the control sample (19.1 ± 2.08 mm). Additionally, Table 3 shows that the effect of Mn x O y nanoparticles on the root length of Hordeum vulgare L. culture has a nonlinear relation with an extremum at 0.05 mg/mL (34.89 ± 2.34mm). The growth of the root length of samples treated with Mn x O y nanoparticles at the point of 0.05 mg/mL is higher by 62.8% (21.43 ± 3.54 mm), 32.7% (26.29 ± 3.22 mm), and 158.9% (13.48 ± 2.66 mm) compared to samples treated with L-methionine, KMnO 4 , and the control sample, respectively. Significant differences from control (group 3) p < 0.01-**, p < 0.001-***, differences from group 1 p < 0.01- § §, p < 0.001- § § §, differences from group 2 p < 0.001-###, differences from group 3 p < 0.01-••, p < 0.001-•••. Differences between groups are considered statistically significant at p <0.05.
Analysis of seed germination energy versus substance concentration showed the predominance of the sample treated with Mn x O y nanoparticles at the point of 0.05 mg/mL (66.00 ± 2.55%) by 50.0% compared to the control sample (44.00 ± 1.78%) [95], by 10.0% compared to the sample treated with L-methionine (60.00 ± 1.00%), and by 13.8% compared to the sample treated with KMnO 4 (58.00 ± 1.58%). The data obtained are consistent with the data of other authors. For example, Esper Neto et al. describes the effect of Mn 2 O 3 nanoparticles on corn seeds. The authors showed that at the concentration of Mn 2 O 3 nanoparticles of 20 mg/L, the increase in root length was 20% and reached 55 mm [96]. An Iincrease in the root length under the influence of nanoparticles was also observed in the work of Landa et al. [57].
The effect of L-methionine-stabilized Mn x O y on the morphological and functional properties of the Hordeum vulgare L. culture is a non-linear dependence (parabolic type) with an extremum at 0.05 mg/mL. (The constructed dependences are presented in the Supplementary (Figures S13-S15). The parabolic type of dependence is due to the fact that at 0.1-5 mg/mL, Mn x O y stabilized with L-methionine is a bound-dispersed system (gel), which makes particle diffusion difficult, and the physiological effect on the seeds of the Hordeum vulgare L. culture is minimal. At concentrations from 0.0005 to 0.1 mg/mL, the samples constitute a free-dispersed system (sol), which is characterized by high biological activity and accessibility. The described process is schematically shown in Figure 16. Nonaggregated Mn x O y nanoparticles stabilized by L-methionine can normalize the level of reception of the essential trace element Mn in the seeds of the culture of Hordeum vulgare L., thereby activating metabolic processes. The obtained tendency meets the results of other scientists [58,59].

Conclusions
In this work, we synthesized of Mn x O y nanoparticles stabilized with L-methionine and studied their structure and phase composition of the material were studied. As a result of quantum chemical modeling, the model of stabilization of Mn x O y with L-methionine was confirmed by IR spectroscopy. The result of the study of physico-chemical properties of the colloidal system Mn x O y -L-methionine was proposed as a gelation mechanism.
The effect of Mn x O y stabilized with L-methionine on the morphofunctional characteristics of seeds of the Hordeum vulgare L. culture is a nonlinear relation with an extremum at 0.05 mg/mL. In our opinion, this effect is related to the correlation of the type of colloidal system of nanoscale Mn x O y with the concentration of Mn x O y particles. Thus, it was shown that at a concentration of Mn x O y < 0.05 mg/mL, the samples are sol-free colloidal systems, and at a concentration of MnxOy > 0.05 mg/mL-gels-cohesive systems.
We found a statistically significant prevalence of seeding length in samples treated with 0.05 mg/mL Mn x O y nanoparticles by 68% compared to the control sample. It is shown that the roots growth length of samples treated with Mn x O y stabilized with L-methionine at the point of 0.05 mg/mL is higher by 62.8%, 32.7%, and 158.9% compared to samples treated with L-methionine, KMnO 4 , and the control sample, respectively. It was found that the germination energy of seeds treated with Mn x O y stabilized with L-methionine at a concentration of 0.05 mg/mL are higher by 50.0% compared to the control sample, by 10.0% compared to the sample treated with L-methionine, and by 13.8% compared to the sample treated with KMnO 4 .
The results of this research allow us to conclude that the use of Mn x O y stabilized with L-methionine in the pre-sowing treatment of unconditioned seeds of agricultural crops with low germination rates will activate its metabolism and thereby increase and improve the growth characteristics of young plants, which is essential for agriculture, especially in extreme conditions with adverse weather conditions and climatic change. It is important to note that the proven effectiveness of Mn x O y stabilized with L-methionine allows them to be used not only on seeds with low germination rates, but also for seeds with high varietal qualities.
The data obtained on the positive effect of Mn x O y stabilized with L-methionine on seeds germination of seeds of the agricultural crop Hordeum vulgare L. formed the basis for further experimental studies on other crops.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/nano13091577/s1, Figure S1. Interaction of the oxygen of the sulfo group of methionine sulfoxide and the hydroxo group of the extreme manganese atom of MnO 2 : model of the molecular complex (a), electron density distribution (b), electron density distribution gradient (c), HOMO molecular orbital (d), LUMO molecular orbital (e), Figure S2. Interaction of the amino group of methionine sulfoxide and the hydroxo group of the extreme manganese atom of MnO 2 : molecular complex model (a), electron density distribution (b), electron density distribution gradient (c), HOMO molecular orbital (d), LUMO molecular orbital (e), Figure S3. Interaction of the carboxyl group of methionine sulfoxide and the hydroxo group of the extreme manganese atom of MnO 2 : model of the molecular complex (a), electron density distribution (b), electron density distribution gradient (c), HOMO molecular orbital (d), LUMO molecular orbital (e), Figure S4  Data Availability Statement: All data are available upon request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.